Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To determine the relationship between the expression of bone proteins and the formation of mineralized-tissue matrix, the biosynthesis of non-collagenous bone proteins was studied in cultures of fetal-rat calvarial cells, which form mineralized nodules of bone-like tissue in the presence of beta-glycerophosphate. The temporal pattern of protein synthesis in both mineralizing and non-mineralizing cultures was studied by metabolic labelling with [35S]methionine, 35SO4(2-) or 32PO4(3-) over a 5-day period. After a 24 h labelling period, the culture media were harvested and the cell layers extracted sequentially with aq. 0.5 M-NH3, followed by 4 M-guanidinium chloride (GdmCl), 0.5 M-EDTA and a second extraction with 4 M-GdmCl. Protein associated with collagenous bone matrix was analysed after digestion with bacterial collagenase. On the basis of [35S]methionine labelling, the major proteins extracted from the mineralizing matrix were secreted phosphoprotein-1 (SPP-1; osteopontin), bone sialoprotein (BSP) and a 14 kDa phosphoprotein. The presence of SPP-1 and BSP in the conditioned media of both mineralizing and non-mineralizing cultures and their incorporation into the mineralizing nodules indicated that these proteins associate with preformed mineral crystals. However, some BSP was also present in GdmCl extracts and, together with a 35 kDa sulphated protein, was released from a bacterial-collagenase digestion of the tissue residue in both non-mineralizing and mineralizing cultures. Two forms of sulphated SPP-1 were identified, a highly phosphorylated 44 kDa species being the predominant form in the mineralized matrix. The BSP was more highly sulphated but less phosphorylated than SPP-1. Bone SPARC (secreted protein, acid and rich in cysteine) protein (osteonectin) was present almost entirely in the conditioned media and did not incorporate 32PO4(3-) or 35SO4(2-). The SPP-1 and the 14 kDa protein were susceptible to thrombin digestion, the 44 kDa SPP-1 being specifically cleaved into 28 and 26 kDa fragments. The fragments were labelled uniformly with [35S]methionine, but the 28 kDa fragment incorporated more 35SO4(2-), but less 32PO4(3-), than the 26 kDa fragment. These studies demonstrate that SPP-1 and BSP are the major osteoblast-derived bone proteins to bind to the bone mineral. That BSP also binds to the collagenous bone matrix indicates a potential role for this protein in linking the hydroxyapatite with collagen.
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PMID:Biosynthesis of bone proteins [SPP-1 (secreted phosphoprotein-1, osteopontin), BSP (bone sialoprotein) and SPARC (osteonectin)] in association with mineralized-tissue formation by fetal-rat calvarial cells in culture. 200 15

Demineralizing extracts of porcine bone contain two large 66-80-kDa sialoproteins and smaller 20- and 23-kDa glycoproteins with similar chemical properties. Each protein was characterized following extraction from fetal calvariae and purification under dissociative conditions using Sepharose CL-6B, followed by fast protein liquid chromatography fractionation on hydroxyapatite and Mono Q resins. Unlike the large sialoproteins, the 20- and 23-kDa glycoproteins did not contain sialic acid. Nevertheless, affinity-purified antibodies raised against the 23-kDa protein recognized both the 20-kDa protein and a 67-kDa sialoprotein on immunoblots. These antibodies also immunoprecipitated a 60-kDa [35S]methionine-labeled protein produced by cell-free synthesis of calvarial bone mRNA, indicating that the smaller proteins were derived from the 67-kDa protein. The two sialoproteins were shown by primary sequence analysis to be secreted phosphoprotein I (SPPI, osteopontin, bone sialoprotein I) and bone sialoprotein (BSP, bone sialoprotein II). The SPPI was also characterized by its susceptibility to thrombin which produced a 23-kDa fragment, similar to the glycoprotein isolated, and a 30-kDa fragment. Amino-terminal sequence analysis of the 23- and 20-kDa proteins revealed that these proteins were derived from the carboxyl-terminal half of the SPPI molecule, the proteins showing 58% identity with human and rat, and 50% identity with mouse, SPPI sequences. Both the 23- and 20-kDa proteins appeared to be generated by the activity of an endogenous trypsin-like protease that cleaves at Arg-Ser (residues 155-156) and Lys-Ala (residues 182-183) bonds. Radiolabeling studies performed in vitro showed that the 23-kDa fragment was detectable in mineralized tissue within 4 h. The fragment was phosphorylated but, unlike SPPI, was not sulfated. The rapid generation of the 23-kDa glycoprotein and its presence in different bone tissues at different developmental stages indicate that the fragmentation of SPPI is important in bone formation and remodeling.
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PMID:Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein I (SPPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein. Demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI. 233 43

The chemical nature of urinary stone protein is poorly understood. We have sequenced a cDNA of urinary calcium oxalate stone protein extracted with EDTA. cDNA sequences showed complete identity between urinary stone protein and human osteopontin. Osteopontin protein was detected by staining with Stains-All, which specifically stains phosphoproteins, and by digestion with the highly specific protease thrombin, demonstrating that urinary calcium oxalate stones consist of osteopontin protein. We used a technique of in situ hybridization to detect osteopontin mRNA in the kidney. In control rats, distal tubular cells were sporadically positive, and proximal tubular cells and glomeruli were negative for osteopontin mRNA. A rat model of stone formation was induced with glyoxylic acid. In stone-forming rats, staining of distal tubular cells was remarkably increased, but proximal tubular cells and glomeruli were still negative. Immunostaining for the osteopontin protein also revealed that epithelial cells of distal tubules were weakly positive in control rats and significantly increased in stone-forming rats, although proximal tubular cells and glomeruli were negative. Northern blot analysis showed a significant increase of osteopontin mRNA in stone-forming rats in proportion to the dosage and the duration of the stone-inducing drugs. These results show that osteopontin in the kidney is presumably involved in urinary stone formation as the stone matrix.
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PMID:Structure and expression of the mRNA encoding urinary stone protein (osteopontin). 832 91

Osteopontin (OPN) has been identified as a matrix protein of calcium oxalate urinary stones by sequencing c-DNA for urinary stone protein. OPN, a phosphoprotein, is susceptible to thrombin digestion and specifically stainable with Stains-All. We examined the matrix in 4 kinds of urinary stones, calcium oxalate dihydrate, calcium phosphate, magnesium ammonium phosphate and uric acid; for the presence of OPN by staining thrombin-digestion and undigested matrix with Stains-All. Matrix was extracted with a 0.1 M ethylenediamine tetraacetic acid (EDTA) solution. Furthermore, the amino acid sequence was determined for the NH2-terminal 20 amino acid residues. OPN was identified in calcium oxalate dihydrate and calcium phosphate stones, but was absent in magnesium ammonium phosphate and uric acid stones. Our findings suggest that OPN, which binds tightly to hydroxyapatite and is related to bone formation as bone matrix, also participates in the formation of calcium-containing urinary stones.
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PMID:Detection of osteopontin as matrix protein in calcium-containing urinary stones. 936 40